Potential application of aminiotic stem cells in veterinary medicine

Abstract In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.


Introduction
Stem cells have a capacity for differentiation and self-renewal. The great interest in science by these cells occur due to its immunomodulatory factors and tissue repair that make possible the use in therapies and innovative treatments (Faita et al., 2016).
The fetal membranes are essential for embryonic development, as they provide maternofetal exchange. It consists of four different membranes: amnion, chorion, the vitelline sac and the allantoic sac (Mossman, 1987). The amnion is the innermost layer of fetal membranes, and contains a thick basement membrane and an avascular stroma (Malak and Bell, 1994) it acts as an additional maternofetal barrier (Leiser and Kaufmann, 1994). Other potential studies involved embryo development and stem cell niches is also highlighted in our data in canines (Martins et al., 2011;Pieri et al., 2015;De Souza et al. 2018).
Amniotic membrane stem cells (AMSC) have previously been described for their immunoregulatory properties (Bossolasco et al., 2006), their differentiate and self-renew potential (Kita et al., 2010) and vigorous cell proliferation. The AMSC are isolated from different species as canine, feline, equine, suine, murine and humans. The application of AMSC occur especially in dogs (Park et al., 2012;Cardoso et al., 2016) and the cats (Vidane et al., 2017), although the murine and suine are good specie models for humans studies.
Thus, this study aimed to carry out amniotic stem cells review about their applicability in the treatment in veterinary medicine.

Stem cells lineages
Discovered by Becker et al. (1963), the stem cells are undifferentiated cells able to perform selfrenewal and differentiate into the most diverse functional cell types (Park et al., 2015;Dai et al., 2016;Sobhani et al., 2017), thus being considered promising sources for the use in tissue engineering and organ regeneration.
Stem cells can be classified according to their potential for differentiation. Totipotent cells are those able to differentiate in all cell lines, including extraembryonic tissues, examples of totipotent cells include the zygote and its early cleavage products. The pluripotent cells are those capable of differentiating only in cells belonging to the three germ layers (Morgani et al., 2013) as embryonic stem cells (ESCs) (Slack, 2018) and induced pluripotent stem cells (iPSCs), somatic cells reprogramed do pluripotency (Takahashi and Yamanaka 2006). The iPSCs were initially produced of cells of mice and humans, however, there is currently reprogramming cells several animal species (Takahashi and Yamanaka, 2006;Takahsshi et al., 2007;Zhang et al., 2015;Gonçalves et al., 2017). Thus, besides the great importance for studies with a therapeutic objective, the IPSCs are also important in veterinary medicine for the maintenance of genetic material of animals with great economic importance (Kumar et al., 2015).
Another type of classification are the multipotent cells, which have ability to differentiate into all cell types within a given lineage (Ratajczak et al., 2014;Khanlarkhani et al, 2016). Although multipotent cells have a lower potential for differentiation, they are considered an important source for cellular therapy (Mirzaei et al., 2018), since they have genetic stability, low immunological profile and slower metabolism when compared to pluripotent stem cells, characteristics that decrease the risk of rejection (Liao and Tse, 2013). A further advantage of multipotent stem cells is the accessibility, and the fact that they can be isolated from the most diverse cell types (Khanlarkhani et al., 2016;Mirzaei et al., 2018). The mesenchymal stem cell (MSC) is a type of multipotent stem cell present in most adult tissues, a heterogeneous cell that plays a key role in the development and renew of organs (Klein, 2016). Several reports have shown that MSCs are commonly isolated and characterized from adipose tissue, bone marrow, umbilical cord, liver, as well a fetal attachments such as yolk sac and amnion (Seo et al., 2009;Uranio et al., 2011;Wenceslau et al., 2011;Reich et al., 2012;Mançanares et al., 2015). This adaptability and easy access make this stem cell a great candidate for use in transplants and therapies.

Mesenchymal stem cells
The MSCs were described by Friedenstein et al. (1970) in bone marrow as a component of the marrow stromal cell population that collectively supports hematopoietic stem cell renew and differentiation (Largeault, 2004;Martins et al., 2007). There are MSC niches in many adult tissues and organs. These cells play an active role in the homeostasis of these sites and can be isolated from the umbilical cord blood and matrix, adipose tissue, synovial membranes and embryonic and extraembryonic tissues.
According to the International Society of Cell Therapy the criteria for the characterization of human MSC are adherence to the plastic, positive expression for the surface markers CD105, CD73 and CD90, negative expression for the markers CD45, CD34, CD14 or CD11b, CD79a or CD19 and the HLA-DR surface molecules, capability to differentiate (osteoblasts, adipocytes and chondrocytes) in vitro (Dominici et al., 2006). According to Casteilla et al. (2011), Tharasanit et al. 2011, Mançanares et al. (2015), MSCs exhibit fibroblastoid morphology in culture.
MSC are easy to isolate, cultivate, and manipulate. These cells have immunoregulation and immunosuppressive characteristics, great plasticity and the potential for therapeutic applications for a variety of clinical conditions (Nardi and Meirelles, 2006;Oliveira et al., 2014;Samsoraj et al., 2017).

Amniotic membrane
The amnion is the most internal extraembrionary membrane, composed of a thin, elastic, translucent and semi-permeable membrane, derived from the ectoderm that covers the fetus and is closely connected to the chorionic membrane (Fernandes et al., 2012;Favaron et al., 2015).
Amniotic membrane (AM) is avascular tissue (Miglino et al., 2006;Dua et al., 2004). It has the function of involving the embryo and delimits the amniotic cavity, which is filled by amniotic liquid to avoid mechanic shocks (Moore and Persaud, 2008;Mamede et al., 2012;Koob et al., 2014).
Basically, the amniotic membrane is composed of two cell types. The epithelial layer composed by the epithelial amniotic cells which present a cuboidal/columnar morphology derived from ectoderm. And the second cell type is composed of mesenchymal stromal cells that are derived from mesoderm. Both populations have similar markers and have the potential to differentiate in vitro in the main mesodermal lineages (Chang et al., 2010;Díaz-Prado et al., 2011).
The AM has particular characteristics such as anti-inflammatory, anti-bacterial, anti-viral and immunological action. Also contains numerous growth factors, cytokines, and signaling molecules that play important roles in fetal development and gestation (Kogan et al., 2018).
Due to the multipotent properties, cells from the amniotic membrane have been investigated for being an attractive source for tissue transplantation (Mamede et al., 2012).

Amniotic Membrane Stem Cells (AMSC)
The AMSC are derived from extraembryonic mesoderm and are randomly distributed in the extracellular matrix of amnio which are rich in collagen and laminin fibers. They are multipotent cells with characteristics that favor their use, such as low immunogenicity and carcinogenicity, production of regulatory molecules of the immune system, high plasticity and propagation in culture and presence of high concentration of lysosomes (Chang et al., 2010;Cremonesi et al., 2011;Vidane et al., 2014;Cardoso et al., 2016;Faita et al., 2016;Magatti et al., 2016;Miki et al., 2016).
The AMSC demonstrates absent immunogenic properties and production of anti-inflammatory and bactericidal substances that provide their use in the treatment of skin wounds, cutaneous ulcers, and ophthalmic disorders such as corneal ulcers (Díaz-Prado et al., 2011;Parolini et al., 2008).
Furthermore, the AMSC are considered excellent sources of stem cells for regenerative therapies of the nervous system due to neural differentiation ability (Uranio et al., 2011;Park et al., 2012).
These cells have advantages over adult stem cells because they preserve embryonic, immunosuppressive and pluripotent characteristics, which are confirmed by the expression of embryonic markers such as OCT-4 and NANOG Vita et al., 2012;Saulnier et al., 2016). Other advantage of AMSC is because they are easy to acquire, offer no damage to donors, and have a lower immune response, making them important in research for regenerative medicine, since inflammation and immunogenicity are crucial factors for successful transplantation (Kim et al. 2014).
The cells derived from the amniotic membrane have the same characteristics of mesenchymal stem cells, according to the International Society of Cell Therapy criteria (Cardoso et al., 2016), and their morphology shows fibroblast characteristics (Fig. 1).

Isolation Protocols for AMSC
There are many types of isolation protocols for AMSC in each species, although they are quite similar. In this review, we will describe the protocols most use with different species in our lab.
For species which the site of collection is more contaminated, as swine and horses, we strongly suggest the refrigeration of samples (1h -2h) before the beginning of the mechanic process, in order to reduce the bacterial activity. So, the amniotic membrane must be mechanically dissect and isolated from embryonic attachments. Under sterile conditions, the tissue must be washed with PBS supplemented with 1% penicillin/streptomycin (P/S, Sigma, USA). After this step, the sample will be transfer to a clean dish and mince with scalpels to become homogeneous and pasty.
After the above-described process, the sample is submitted to chemical digestion using collagenase type I (Sigma, USA) which the concentration and time are described below to each species (Tab. 1).
After digestion the collagenase enzyme has to be neutralizing using the same volume of expansion media to the cell suspension. So, the digested sample is centrifuged at 1600 rpm/5 min, and then cell pellet is obtained to be culture with the expansion media and keep at 37°C and 5% CO2.

Neurologic applications
The AMSC is considered a source of election to treat spinal cord injury. Sankar et al. (2003), showed good interaction of human amniotic membrane stem cells when applied in iatrogenic spinal cord injury in monkeys, and they observed a significant remyelination, as well as their ability to modulate the glial scar. And a similar study was performed by Zhi-Yuan et al. (2006) who observed a returning of motor function in the hind limbs of treated animals. Furthermore, Meng et al. (2008) studied the co-transplantation of AMSC with stem cells of neuronal origin in rats with chronic spinal cord injury. And again, the results demonstrated a significant locomotor improvement in addition to neuronal survival and differentiation. Kakishita et al. (2003) verified that human amniotic epithelial cells secrete biologically active neurotrophins and can enhance the survival of dopamine neurons, being a promising therapeutic tool in Parkinson's disease. As well as Yang et al. (2009) who also observed a increasing of dopamine and its metabolic products in the striatum in rats with Parkinson's disease.

Ischemic dysfunction
Kim and Choi (2011) observed neovascularization in mouses suffering of limb ischemia. They administered intramuscularly hAMSCs on the leg whose femoral vessels were ligated. The blood flow recovery was significantly higher in the transplanted group, when compared with the control group, being an attractive source for the treatment of ischemic diseases.
And Tao et al. (2012) transplanted human amniotic membrane stem cells transfected with the brain derived neurotrophic factor (BDNF) into the brains of rats with induced stroke. This technique ameliorated the behavioral dysfunction and reduced the infarct volume, improving functional recovery.

Musculoskeletal disorders
The stem cell therapy is a routine treatment in horses, and the amnion plays an important role in this scenario. In mares with endometriosis, the AMSC cells were used because of their potential to improve cell replenishment based on gene expression profiling when low proliferation of uterine cells is associated to pregnancy failure in an in vivo study, showing lower rate of reinjury and faster resume to their activities (Corradetti et al., 2014). In tendon repair, the re-injury was lower in treatment with AMSC than compared with BM-MSC ascertained by ultrasonography (Lange-Consiglio et al., 2013b). Still, biomaterials composed for amniotic membrane has attracted attention. They have being associated to better mechanical properties in tendons injuries, plus this membrane has been used to accelerate healing wounds (Violini et al., 2012;Hortensius et al., 2016).

Others applications
A promising use of large animal models must be done before clinical trial and confirm efficacy of therapy and controversial results must be followed (Gonçalves et al, 2014). Kamiya et al. (2005) showed efficacy in suppressing corneal inflammatory reactions, when topically applied human amniotic membrane culture supernatant in mices with induced corneal neovascularization. Vidane et al. (2017) evaluated the effects of intravenous administration of allogeneic feline amniotic membrane stem cells in cats with naturally occurring chronic kidney disease. Despite the kidney architecture and morphology did not change during the treatment, the tranplantation showed renoprotective effect, improved renal function, delaying the progression of the disease and stabilizing the clinical condition of the animals.
And finally, the AMSC are described into application in humans to aid in treatment of cutaneous wounds, burns, and superficial ocular reconstruction (Ward and Bennett, 1984;Avila et al., 2001;Gomes et al., 2005).